Project Summary Walking is an essential and conserved behavior across the animal kingdom. The ability to move in a coordinated, robust yet flexible manner is a crucial for an animal?s survival in the ever changing environment. Motor behaviors are controlled by sensory neuropils in the brain which talk to motor centers such as the ventral nerve cord (VNC) in the thorax to execute precise motor outputs. Descending neurons are the primary conduits of information that accomplish this connection between the brain and the VNC. However the downstream target neurons and complex circuits that precisely govern the execution of a specific kind of limbed motor output such as running, turning, slowing down, switching gait, climbing, stepping etc remain individually undefined. This project aims to map these individual circuits that control different walking kinematics. In aim 1, we will map the post-synaptic targets of descending neurons using a translabeling technique called Trans Tango. We already know from previous studies that have characterized the morphology of all the descending neurons in Drosophila, which DNs target the leg neuropils in the ventral nerve chord. These descending neurons are very likely to be directly implicated in controlling walking behavior. Additionally, we also have broad behavioral information for a subset of the DNs. Using this existing morphological and behavioral data, we can start to map out the motor and premotor circuits that are downstream of these DNs and reveal the distinct circuit components that control specific walking kinematics. Some of the DNs we plan to screen, affect similar behaviors hence it will be interesting to reveal whether they converge onto similar or different targets. In the second aim, we aim to use a computational approach search for generation Gal4 lines whose neuronal expression patterns match the downstream target neurons we want to study. Existing split Gal4 lines will be identified or new ones will be generated in order to gain genetic access to the downstream circuit neurons of interest. In the third aim, we will use three behavior setups: Flywalker, the Arena and the fly-on-ball setup to study a range of walking parameters in high resolution using optogenetics intersectional strategies. We will use optogenetic activation, silencing and also epistasis experiments to map out which components of the circuit are sufficient or necessary for a particular walking kinematic.